Expression of P450 2C9 mutants
P450 2C9 mutants were coexpressed in bicistronic format with hNPR in E. coli cotransformed with the expression vector pGro7 encoding the bacterial chaperones GroES and GroEL. Values represent the mean of two to four separate expression trials.
CYP2C9 Form | P450 Hemoprotein Expression | hNPR Expressiona | |
|---|---|---|---|
| nmol l-1 culture | |||
| 2C9*1 (wild type) | 144 | 207 | |
| R97A | <1 | 190 | |
| R97K | 7 | 195 | |
| R97E | <1 | 187 | |
| R97Q | <1b | 205 | |
| R97V | <1 | 230 | |
| R97L | <1 | 213 | |
| R97P | 15 | 91 | |
| R97I | 16 | 100 | |
| R97T | <1 | 188 | |
| K72E | 193 | 181 | |
| K72V | 147 | 130 | |
| K72Q | 244 | 177 | |
| K72L | 169 | 71 | |
| (hNPR monocistronic) |
| 484 | |
↵ a Yields of hNPR were calculated assuming a specific activity of 3200 nmol of cytochrome c reduced min-1 nmol hNPR-1 (Yasukochi and Masters, 1976)
↵ b A weak P450 spectrum was detected for this mutant, but the expression level was too low to be quantified